General considerations for selecting optimal binding conditions for Proteus IMAC kits

This kit exploits the hexahistidine sequence that permits efficient purification of the expressed protein from a broad host such as bacterial cells, Baculovirus vectors, mammalian cells or yeast. Baculovirus, mammalian cells and yeast expression vectors are often used to express eukaryotic proteins as they generate proteins with the similar posttranslational modifications such as phosphorylations and glycosylations.

Lysis conditions, such as the nature of the lysis buffer, depend upon the type of expression vector. Mammalian or Baculovirus-infected insect cells can be lysed by sonication at +4 ºC with either freeze/thaw cycles or addition of up to 1 % non-ionic detergents and cell lysis of E.coli is usually achieved by sonication on ice or homogenization either with or without lysozyme treatment.

The culture pellet is resuspended in lysis buffer at a pH close to pH 7.4-8.0 using a similar concentration of buffer, imidazole and NaCl to that of a pre-equilibration buffer used for metal chelate chromatography. Binding of His-tagged soluble proteins present in the cytoplasm or periplasm and insoluble aggregates in the presence of denaturants occurs close to physiological pH.

Typically, a protease inhibitor cocktail, such as Boehringer "Complete EDTA-free", 5-50 µg/ml DNase I and 10 mM β-mercaptoethanol are added to the lysis buffer. Addition of β-mercaptoethanol to the lysis buffer and the binding, wash and elution buffers are optional. Its inclusion depends upon whether the His-tagged protein elutes with contaminants as β-mercaptoethanol can reduce all disulphide bonds formed between the contaminating proteins and the target protein. Initially, the researcher should try to bind the His-tagged protein directly from the cleared lysate.

It is imperative that the lysate is completely clear as any particulate matter e.g. cell debris will partially foul the resin and cause spin times for the binding, washing and elution steps to be increased. It is important that the sample is first filtered through a 0.2 µm syringe-end filter to remove particulates that could clog the resin flow channels. All samples should be filtered just prior to loading even if they have been filtered several days before the chromatographic run.

If the binding efficiency is poor and the lysis buffer differs significantly from the pre-equilibration buffer, optimal binding of the His-tagged protein to the Ni-IDA spin columns can be achieved by rapid dialysis, diafiltration using ultrafiltration concentrators, gel-filtration chromatography in the appropriate pre-equilibration buffer or titration with a concentrated stock solution of pre-equilibration buffer.

Note that the precise conditions for binding, washing and eluting your target protein may need to be optimized empirically as there are several factors such as accessibility of the His-tag which affect protein behaviour in non-denaturing conditions during metal chelate chromatography.

Aggregation/precipitation of proteins is common during storage and repeated freeze/thaw cycles. Of equal importance is the ability to process the samples rapidly and, if the need arises, to be able to purify the target protein in a Proteus spin column at 4 °C.

Optimal conditions for binding the target molecule to a resin are critical for successful separation of the protein. If the binding conditions are not optimal with respect to pH, salt concentration, presence of metal ions or chelating agents, flow rates, residence time etc, purification is adversely affected.